A Protocol for Agrobacterium-mediated Transformation of Cucumber (Cucumis sativus L.) from cotyledon explants

X, Li; Ma, Si; Shan, Nan; Zhang, Xiaolan; Sui, Xiaolei; Zhang, Zhenxian

doi:10.1038/protex.2017.107

Cited by 18 publications

(21 citation statements)

References 37 publications

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“…To generate overexpression LL transgenic cucumber lines, the CaMV35S:LL construct was delivered into Agrobacterium tumefaciens strain AGL1, which was transformed into the cucumber line Poinsett 76 using cotyledons as explants following Li et al (2017) with minor modifications. In brief, the dissected cotyledons were incubated with the Agrobacterium culture (OD 600 = 0.7) for 12 min facilitated with vacuum infiltration.…”

Section: Expression Of Ll Candidate Genes In Arabidopsis and Cucumbermentioning

confidence: 99%

LITTLELEAF (LL) encodes a WD40 repeat domain‐containing protein associated with organ size variation in cucumber

et al. 2018

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Plants employ tight genetic control to integrate intrinsic growth signals and environmental cues to enable organs to grow to a defined size. Many genes contributing to cell proliferation and/or cell expansion, and consequently organ size control, have been identified, but the regulatory pathways are poorly understood. Here we have characterized a cucumber littleleaf (ll) mutant which exhibits smaller organ sizes but more lateral branches than the wild type. The small organ size in ll was due to a reduction of both cell number and cell size. Quantitative trait locus (QTL) analyses revealed co-localization of major-effect QTLs for fruit size, fruit and seed weight, as well as number of lateral branches, with the LL locus indicating pleiotropic effects of the ll mutation. We demonstrate that LL is an ortholog of Arabidopsis STERILE APETALA (SAP) encoding a WD40 repeat domain-containing protein; the mutant protein differed from the wild type by a single amino acid substitution (W264G) in the second WD40 repeat. W264 was conserved in 34 vascular plant genomes examined. Phylogenetic analysis suggested that LL originated before the emergence of flowering plants but was lost in the grass genome lineage. The function of LL in organ size control was confirmed by its overexpression in transgenic cucumbers and ectopic expression in Arabidopsis. Transcriptome profiling in LL and ll bulks revealed a complex regulatory network for LL-mediated organ size variation that involves several known organ size regulators and associated pathways. The data support LL as an important player in organ size control and lateral branch development in cucumber.

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Section: Expression Of Ll Candidate Genes In Arabidopsis and Cucumbermentioning

confidence: 99%

LITTLELEAF (LL) encodes a WD40 repeat domain‐containing protein associated with organ size variation in cucumber

et al. 2018

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“…However, cucumber is one of the most difficult species to transform, although it has been carried out for 39 years [7]. The highest transformation efficiency of cucumber was 26% [2], while the lowest was only 0.1%, and most reports are between 1% and 10% [2]. Moreover, there have been few reports about the transformation of cucumber 'Xintaimici' [39].…”

Section: Discussionmentioning

confidence: 99%

Improved Agrobacterium Tumefaciens-Mediated Transformation of Cucumber via Modified use of Antibiotics and Acetosyringone

Chai

Du²,

Fan

et al. 2020

Preprint

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Abstract Background: Cucumber (Cucumis sativus) is one of the most important vegetable crops in the world. As conventional breeding of cucumber is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced stress resistance and nutritional value. However, the efficiency of the transformation system depends on genotypes, transformation conditions, selection agents, etc. This study aims to speed up the process of Agrobacterium-mediated transformation of cucumber. ‘Xintaimici’, a very popular and typical north China-type cucumber variety, was transformed with Agrobacterium GV3101. The strain carried the pCAMBIA2300s plasmid, a double vector with the marker gene neomycin phosphotransferase II (npt II). Results: The research results indicated that cefotaxime sodium was suitable for inhibiting Agrobacterium in the screening and bud elongation stages. Timentin was best used during the rooting stage. Furthermore, 25 mg/L kanamycin was used in the early stage of screening and increased to 50 mg/L for further screening. At the bud elongation and rooting stages, 75 and 100 mg/L kanamycin was used, respectively, to improve the screening efficiency. To obtain the highest regeneration frequency of resistant buds, 50, 150, and 100 μM acetosyringone was added in the pre-culture medium, infection solution, and co-culture medium, respectively. To confirm the presence of the transgenes, DNA from npt II transgenic cucumber plants was analysed by polymerase chain reaction after transplanting resistant regenerated plants. Conclusions: We finally achieved an 8.1% conversion, which is among the highest values reported to date using the cucumber ‘Xintaimici’. Thus, an effective protocol for Agrobacterium tumefaciens-mediated genetic transformation of cucumber was optimized.

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“…But cucumber is still one of the most difficult species for transformation, although it has been carried out for 39 years [7]. The highest transformation efficiency of cucumber was 26% [2], while the lowest was only 0.1%, and mostly between 1%-10% [2]. Moreover, there were few reports about the transformation of cucumber 'Xintai mici' [39].…”

Section: Discussionmentioning

confidence: 99%

“…Cucumber (Cucumis sativus) is one of the most important vegetable crops widely grown in the world. The studies on gene function and genetic breeding of cucumber have been concerned world widely [1,2]. Abnormal expressions of genes in transgenic cucumber plants can help us to engineer and select more robust crop species, such as resistance to biotic and abiotic stress, fruit quality improvement [3], growth and development [4,5].…”

Section: Introductionmentioning

confidence: 99%

Improved the Agrobacterium tumefaciens-mediated transformation of cucumber by a modified the using of antibiotics and acetosyringone

Chai¹,

Du²,

Fan³

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: Cucumber (Cucumis sativus) is one of the most important vegetable crops in the world. As conventional breeding of cucumber is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced stress resistance and nutritional value. However, the efficiency of the transformation system depends on genotypes, transformation conditions, selection agents, etc. This study aims to speed up the process of Agrobacterium-mediated transformation of cucumber. ‘ Xintai mici ’, a very popular and typical north China-type cucumber variety, was transformed with Agrobacterium GV3101. The strain carried pCAMBIA2300s plasmid, a double vector with the marker gene of neomycin phosphotransferase II ( npt II). Results: The research results indicated that cefotaxime sodium was suitable for inhibiting Agrobacterium in the stage of screening and bud elongation. Timentin was best used during rooting stage. Furthermore, 25 mg/L kanamycin was used in the early stage of screening and increased to 50 mg/L for further screening. At the bud elongation and rooting stage, 75 and 100 mg/L kanamycin was used respectively to improve the screening efficiency. In order to obtain the highest regeneration frequency of resistant buds, 50, 150, and 100 μM acetosyringone were added in the pre-culture medium, infection solution, and co-culture medium respectively. To confirm the presence of the transgenes, DNA from npt II transgenic cucumber plants was analyzed by polymerase chain reaction after transplanting resistant regenerated plants. Conclusions: We finally achieved an 8.1% conversion, which was among the highest values reported until date using cucumber ‘ Xintai mici ’. Thus an effective protocol for Agrobacterium tumefaciens -mediated genetic transformation of cucumber was optimized.

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A Protocol for Agrobacterium-mediated Transformation of Cucumber (Cucumis sativus L.) from cotyledon explants

Cited by 18 publications

References 37 publications

LITTLELEAF (LL) encodes a WD40 repeat domain‐containing protein associated with organ size variation in cucumber

LITTLELEAF (LL) encodes a WD40 repeat domain‐containing protein associated with organ size variation in cucumber

Improved Agrobacterium Tumefaciens-Mediated Transformation of Cucumber via Modified use of Antibiotics and Acetosyringone

Improved the Agrobacterium tumefaciens-mediated transformation of cucumber by a modified the using of antibiotics and acetosyringone

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